scholarly journals 145 Comparison of CAR-T cell manufacturing platforms reveals distinct phenotypic and transcriptional profiles

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A153-A153
Author(s):  
Hannah Song ◽  
Lipei Shao ◽  
Michaela Prochazkova ◽  
Adam Cheuk ◽  
Ping Jin ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Effector Memory ◽  
Cell Phenotype ◽  
Gene Clustering ◽  
Car T Cells ◽  
Car T Cell ◽  
Cell Manufacturing ◽  
Car T ◽  
And Function

BackgroundWith the clinical success of chimeric antigen receptor (CAR)-T cells against hematological malignancies, investigators are looking to expand CAR-T therapies to new tumor targets and patient populations. To support translation to the clinic, a variety of cell manufacturing platforms have been developed to scale manufacturing capacity while using closed and/or automated systems. Such platforms are particularly useful for solid tumor targets, which typically require higher CAR-T cell doses that can number in the billions. Although T cell phenotype and function are key attributes that often correlate with therapeutic efficacy, it is currently unknown whether the manufacturing platform itself significantly influences the output T cell phenotype and function.MethodsStatic bag culture was compared with 3 widely-used commercial CAR-T manufacturing platforms (Miltenyi CliniMACS Prodigy, Cytiva Xuri W25 rocking platform, and Wilson-Wolf G-Rex gas-permeable bioreactor) to generate CAR-T cells against FGFR4, a promising target for pediatric sarcoma. Selected CD4+CD8+ cells were stimulated with Miltenyi TransAct, transduced with lentiviral vector, and cultured out to 14 days in TexMACS media with serum and IL2.ResultsAs expected, there were significant differences in overall expansion, with bag cultures yielding the greatest fold-expansion while the Prodigy had the lowest (481-fold vs. 84-fold, respectively; G-Rex=175-fold; Xuri=127-fold; average of N=4 donors). Interestingly, we also observed considerable differences in CAR-T phenotype. The Prodigy had the highest percentage of CD45RA+CCR7+ stem/central memory (Tscm)-like cells at 46%, while the bag and G-Rex cultures had the lowest at 16% and 13%, respectively (average N=4 donors). In contrast, the bag, G-Rex, and Xuri cultures were enriched for CD45RO+CCR7- effector memory cells and also had higher expression of exhaustion markers PD1 and LAG3. Gene clustering analysis using a CAR-T panel of 780 genes revealed clusters of genes enriched in Prodigy/de-enriched in bag, and vice versa. We are currently in the process of evaluating T cell function.ConclusionsThis is the first study to our knowledge to benchmark these widely-used bioreactor systems in terms of cellular output, demonstrating that variables inherent to each platform (such as such as nutrient availability, gas exchange, and shear force) significantly influence the final CAR-T cell product. Whether enrichment of Tscm-like cells in the final infusion product correlates with response rate, as has been demonstrated in the setting of CD19 CAR-Ts, remains to be seen and may differ for FGFR4 CAR-Ts and other solid tumors. Overall, our study outlines methods to identify the optimal manufacturing process for future CAR-T cell therapies.

scholarly journals Enhanced CAR-T activity against established tumors by polarizing human T cells to secrete interleukin-9

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Lintao Liu ◽  
Enguang Bi ◽  
Xingzhe Ma ◽  
Wei Xiong ◽  
Jianfei Qian ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Antitumor Activity ◽  
Effector Memory ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Ifn Γ

AbstractCAR-T cell therapy is effective for hematologic malignancies. However, considerable numbers of patients relapse after the treatment, partially due to poor expansion and limited persistence of CAR-T cells in vivo. Here, we demonstrate that human CAR-T cells polarized and expanded under a Th9-culture condition (T9 CAR-T) have an enhanced antitumor activity against established tumors. Compared to IL2-polarized (T1) cells, T9 CAR-T cells secrete IL9 but little IFN-γ, express central memory phenotype and lower levels of exhaustion markers, and display robust proliferative capacity. Consequently, T9 CAR-T cells mediate a greater antitumor activity than T1 CAR-T cells against established hematologic and solid tumors in vivo. After transfer, T9 CAR-T cells migrate effectively to tumors, differentiate to IFN-γ and granzyme-B secreting effector memory T cells but remain as long-lived and hyperproliferative T cells. Our findings are important for the improvement of CAR-T cell-based immunotherapy for human cancers.

scholarly journals Third-Generation CAR T Cells Targeting CD19 Are Associated with an Excellent Safety Profile and Might Improve Persistence of CAR T Cells in Treated Patients

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 51-51 ◽  
Author(s):  
Maria-Luisa Schubert ◽  
Anita Schmitt ◽  
Brigitte Neuber ◽  
Angela Hückelhoven-Krauss ◽  
Alexander Kunz ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
University Hospital ◽  
Third Generation ◽  
Advisory Committees ◽  
Car T Cells ◽  
Car T Cell ◽  
Cell Manufacturing ◽  
Car T

Introduction T cells transduced with a chimeric antigen receptor (CAR) have demonstrated significant clinical efficacy in patients with lymphoid malignancies including relapsed or refractory (r/r) B-lineage acute lymphoblastic leukemia (ALL) or r/r B-cell non-Hodgkin's lymphoma (NHL). Second-generation CAR T cells comprising 4-1BB or CD28 as costimulatory domains have become commercially available for the treatment of patients with CD19+ lymphoid malignancies. However, achievement of durable clinical responses remains a challenge in CAR T cell therapy. Consequently, third-generation CARs incorporating both elements might display short-term efficacy with potent and rapid tumor elimination (CD28) as well as long-term persistence (4-1BB). So far, only two clinical trials employing third-generation CAR T cells have been reported. Both enrolled 31 patients in summary and demonstrated favorable results for third-generation CAR T cells. Here, we report on first results of our investigator-initiated trial (IIT) on third-generation CD19-directed CAR T cells: The Heidelberg CAR trial 1 (HD-CAR-1; NCT03676504; EudraCT 2016-004808-60) is a phase I/II trial initiated in September 2018 with in-house leukapheresis and CAR T cell manufacturing in full compliance with European Good Manufacturing Practice (GMP) guidelines at the University Hospital Heidelberg. Methods Adult and pediatric patients with r/r ALL and patients with r/r chronic lymphocytic leukemia (CLL) or NHL including diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL) or mantle cell lymphoma (MCL) are treated with autologous T lymphocytes transduced with a CD19 targeting third-generation CAR retroviral vector (RV-SFG.CD19.CD28.4-1BBzeta). The main purpose of HD-CAR-1 is to evaluate safety and feasibility of escalating third-generation CAR T cell doses (1-20×106 transduced cells/m2) after lymphodepletion with fludarabine (30 mg/m2/d on days -4 to -2) cyclophosphamide (500 mg/m2/d on days -4 to -2). Patients are monitored for cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS) and/or other toxicities. In vivo function, survival and anti-tumor efficacy of CAR T cells are assessed. Results To date, 10 patients (3 adult ALL, 2 CLL, 2 MCL, 2 DLBCL, 1 transformed FL) have been enrolled and subjected to leukapheresis. Transduction efficiency of T lymphocytes ranged between 33%-66% and high numbers of transduced CAR T cells were harvested (70-123x106 CAR T cells). No production failure occurred. CAR T cell products were sterile and free from mycoplasma and endotoxins. The copy number per CAR T cell did not exceed 7. Eight patients (2 adult ALL, 2 CLL, 1 MCL, 2 DLBCL, 1 transformed FL) have received the CAR T cell product (6 patients: 106 transduced cells/m2; 2 patients 5×106 transduced cells/m2). No signs of CRS or ICANS > grade 2 have been observed. Only one patient required tocilizumab. No neurological side-effects occurred, even not in patients with involvement of the central nervous system (CNS). In quantitative real-time PCR, CAR T cells were detectable in the peripheral blood (PB) in 3 of 4 analyzed patients or the cerebrospinal fluid (CSF) of an ALL patient with CNS involvement. The CAR T cell frequency reached up to 200,000 copies/µg DNA, in some patients beyond end-of-study at day 90 after CAR T cell administration. Clinical responses to treatment were observed in 6/8 (75%) treated patients so far (2/8 patients have received CAR T cells recently and are not yet evaluable for response). Conclusion Leukapheresis and CAR T cell manufacturing were effective for all patients enrolled in the HD-CAR trial to date. Patients responded clinically to treatment despite low numbers of administered CAR T cells. CAR T cells displayed an excellent safety profile and were detectable for more than 3 months following administration. Furthermore, CAR T cells migrated into different compartments including the CSF in case of CNS involvement. For HD-CAR-1 leukapheresis, CAR T cell manufacturing, CAR T cell administration, patient monitoring and follow-up are performed in-house, providing autarky from transport or production sites outside the University Hospital Heidelberg. Altogether, HD-CAR-1 accounts to clinical evaluation of third-generation CAR T cells that might contribute to long-term CAR T cell persistence, hence improving efficient and durable responses in treated patients. Disclosures Schmitt: Therakos Mallinckrodt: Other: Financial Support . Sellner:Takeda: Employment. Müller-Tidow:MSD: Membership on an entity's Board of Directors or advisory committees. Dreger:AbbVie, AstraZeneca, Gilead, Janssen, Novartis, Riemser, Roche: Consultancy; AbbVie, Gilead, Novartis, Riemser, Roche: Speakers Bureau; Neovii, Riemser: Research Funding; MSD: Membership on an entity's Board of Directors or advisory committees, Other: Sponsoring of Symposia. Schmitt:Therakos Mallinckrodt: Other: Financial Support; MSD: Membership on an entity's Board of Directors or advisory committees, Other: Sponsoring of Symposia.

scholarly journals Cytolytic Activity of CAR T Cells and Maintenance of Their CD4+ Subset Is Critical for Optimal Antitumor Activity in Preclinical Solid Tumor Models

Cancers ◽  
2021 ◽  
Vol 13 (17) ◽  
pp. 4301
Author(s):  
Marianna Csaplár ◽  
János Szöllősi ◽  
Stephen Gottschalk ◽  
György Vereb ◽  
Árpád Szöőr
Keyword(s):  
T Cells ◽  
T Cell ◽  
Antitumor Activity ◽  
Cytolytic Activity ◽  
Effector Memory ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Differentiation Status

Correlative studies of clinical studies for hematological malignancies have implicated that less differentiated, CD8+-dominant CAR T cell products have greater antitumor activity. Here, we have investigated whether the differentiation status of CAR T cell products affects their antitumor activity in preclinical models of solid tumors. We explored if different activation/expansion protocols, as well as different co-stimulatory domains in the CAR construct, influence the short- and long-term efficacy of CAR T cells against HER2-positive tumors. We generated T cell products that range from the most differentiated (CD28.z; OKT3-antiCD28/RPMI expansion) to the least differentiated (41BB.z; OKT3-RetroNectin/LymphoONE expansion), as judged by cell surface expression of the differentiation markers CCR7 and CD45RA. While the effect of differentiation status was variable with regard to antigen-specific cytokine production, the most differentiated CD28.z CAR T cell products, which were enriched in effector memory T cells, had the greatest target-specific cytolytic activity in vitro. These products also had a greater proliferative capacity and maintained CD4+ T cells upon repeated stimulation in vitro. In vivo, differentiated CD28.z CAR T cells also had the greatest antitumor activity, resulting in complete response. Our results highlight that it is critical to optimize CAR T cell production and that optimal product characteristics might depend on the targeted antigen and/or cancer.

CAR-T Cell Production Using the Clinimacs® Prodigy System

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5724-5724 ◽  
Author(s):  
Fenlu Zhu ◽  
Nirav N. Shah ◽  
Huiqing Xu ◽  
Dina Schneider ◽  
Rimas Orentas ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Third Party ◽  
Clinical Use ◽  
Cd8 Cells ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Equity Ownership ◽  
And Function

Abstract Introduction Chimeric Antigen Receptor T (CAR-T) cells redirected against tumor antigens are an effective therapy for B cell malignancies refractory to standard treatments. The production of patient-derived CAR-T cells is complicated and thus far is limited to institutions with experienced researchers and expensive GMP facilities, or to those invited to participate in industry sponsored clinical trials. The outsourcing of CAR T-cell production to third party vendors where cells are collected locally, shipped to the manufacturing site, and then sent back to the institution for infusion can be both costly and timely. As a result, CAR-T cell therapies are not widely available and only patients with means to travel to participating sites and with disease that is stable enough to wait the 2-3 months needed to collect and produce CAR-T cells are eligible for these treatments. At our instution we have explored the use of the CliniMACS® Prodigy (Miltenyi Biotec, Inc) for the production of CAR-T cells. The CliniMACS® Prodigy is an automated device that can be used for cell processing within a closed GMP-compliant system. Using the CAR-T system that includes software, specialized tubing sets, and optimized reagents we demonstrate the processing of CAR-T cells, with similar characteristics to those produced in a more traditional manner, in a closed system that is suitable for clinical use without the need for a clean room manufacturing facility. Methods In collaboration with Miltenyi Biotec, we obtained pre-release and final versions of the CliniMACS® Prodigy TCT process software and the TS520 tubing set that allows for cell enrichment, transduction, wash steps, and expansion all within a single set. Starting material was MNC cells recovered from a leukoreduction system chamber (LRSC) used during platelet collections by apheresis. Materials and reagents included MACS CD4 & CD8 reagents for cell enrichment, TransAct CD3/CD28 reagent for activation, lentiviral CD8 TM-41BB-CD3 zeta-cfrag vectors with either CD19 or CD20/CD19 Ab chains (Lentigen Technology Inc., A Miltenyi Biotec Company), TexMACS culture medium-3% HS-IL2, and PBS/EDTA buffer for wash steps. For two experiments, cells after CD4/CD8 enrichment were activated and transduced in 6 well plates and expanded after day 5 in G-Rex gas permerable devices. Total time for line preparation was 14±1 days. Transduction was measured by Protein L expression using flow cytometry. Line function was measured in 51Cr Release assays and by intracellular cytokine production. Results Starting cells were washed free of platelets and enriched for CD4+ and CD8+ cells using the Prodigy device. We achieved consistent high levels purity (99±3%) and good recovery (51.0±6%) of CD4+ and CD8+ cells (N=5). The enriched cells were 90±12% CD3+. The approximately 10% non-T cells were CD8+ NK cells, that were largely eliminated after cell activation through CD3/CD28 and expansion. A controlled number of 1 x 10E8 cells enriched for CD4+ plus CD8+ cells were retained in the Prodigy and in 2 experiments a smaller fraction of cells was cultured in 6 well plates for activation and initial transduction. Three preparations were conducted in the Prodigy, one using the CD19 vector and two with the CD19+CD20 vector. Transduction efficiency ranged from 21%-46% of total T cells with a modest preference for CD4+ cells. Expansion ranged from 26-40 fold and all of the lines recognized CD19 and/or CD20 targets based on 51Cr release assays or IFN-gamma production. The paired lines generated on the Prodigy versus manual methods showed similar overall transduction, phenotype, and function as shown in the figure for one representative preparation. Conclusions CAR-T cells generated in the Prodigy were similar to those prepared using manual methods in both phenotype and function. This process is timely, requiring 14 days for generation of the target CAR-T cell dose, and does not require outsourcing to third party vendors. All of the Prodigy CAR-T cell preparations met criteria for clinical use in our upcoming Phase I clinical trial. The ability to produce CAR-T cells suitable for clinical use in an entirely closed system without the need for a clean room should allow more centers and patients access to this novel form of immunotherapy. Disclosures Shah: Oncosec: Equity Ownership; Exelixis: Equity Ownership; Geron: Equity Ownership. Orentas:Lentigen Technology, Inc.: Employment. Dropulic:Lentigen Technology Inc. A Miltenyi Biotec Company: Employment. Hari:Merck: Research Funding; BMS: Honoraria.

scholarly journals A BCL2L1 Armoured BCMA Targeting CAR T Cell to Overcome Exhaustion and Enhance Persistence in Multiple Myeloma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 327-327
Author(s):  
Ranjan Maity ◽  
Sacha Benaoudia ◽  
Franz Zemp ◽  
Holly Lee ◽  
Elie Barakat ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Memory T Cells ◽  
Antigenic Stimulation ◽  
T Cell Subsets ◽  
Effector Memory ◽  
Car T Cells ◽  
Car T Cell ◽  
Chronic Antigenic Stimulation ◽  
Car T

Abstract Chimeric antigen receptor (CAR) T cells targeting the B-cell maturation antigen (BCMA) have resulted in deep responses in patients with relapsed MM however most remissions are not sustained. While cellular and molecular mediators of relapse post CAR T therapy in MM are not fully delineated, current data suggest three possible mechanisms including the lack of persistence of the CAR T cell product, acquired exhaustion and less commonly loss of BCMA expression. Using CITE-seq we measured the expansion of variable T cell subsets, T cell specific activation and inhibitor markers and their functional states in serial blood and marrow samples (n=10) collected from patients treated with BCMA targeting CAR T cells. CAR T cells were identified by the expression of the chimeric CAR T cell transcript. With the exception of one patient where biallelic loss of BCMA was identified at relapse, CAR T cells of resistant patients were enriched with terminally exhausted CD45RA+ cells with loss of CD28, low BCL2L1 (gene encoding BCL-XL) expression, high CD57 with co-expression of checkpoint inhibitors (LAG3, TIGIT and PD1). The lack of persistence of the CAR T cells product was notable in all relapsing patients consistent with an activation induced cells death (AICD) specially in the setting of chronic antigenic stimulation. Cognizant of the role BCL-XL plays in T cells survival in response to CD28 co-stimulatory signaling, we postulated that increasing BCL-XL expression is a feasible strategy to enhance CAR T cell resistant to AICD, improve their persistence and anti-BCMA reactivity. To this goal, we designed a 2nd generation lentiviral CAR construct where the anti-BCAM scFV-41BBz CAR and the BCL2L1 cDNA were linked with self-cleaving 2A sequence. The efficiency in eradicating MM cells of this BCL-XL armored CAR (BCMA_BCL2L1_CAR) was compared to that of non-unarmored CAR (BCMA_CAR) in vitro and i n vivo studies. While BCMA_BCL2L1_CAR and BCMA_CAR were equally cytotoxic to OPM2 MM cells, in MM cell lines expressing the FAS death receptor ligand FASLG (MM1S, OCMY5 and H929) BCMA_BCL2L1_CAR viability and cytolytic activity was significantly superior to that of unarmored BCMA_CAR. Of note, the expression of FASLG, a known interferon response gene, was upregulated in H929 cells when co-cultured with CAR T cells. Importantly, under chronic antigenic stimulation conditions (FIG 1A), where CAR T cells were stimulated every 6 days over a 28 days period with irradiated OPM2 cells, we found no phenotypic difference between BCMA_BCL2L1_CAR and BCMA_CAR with respect to the composition of effector memory T cells (Tem: CCR7− CD45RO+ CD45RA−) or central memory T cells (Tcm: CCR7+CD45RO+CD45RA−) or terminal effector / exhausted T cells. However, under these chronic antigenic stimulation conditions, the CAR T cells viability, proliferation (FIG 1B) and anti-MM cytotoxic activities (FIG 1C) of the BCMA_CAR were dramatically reduced compared to that of the BCL2L1 armored CAR. Furthermore, in initial animal studies where NOD-SCID mice were tail vein injected with 2e6 OPM2 MM cells transduced with a luciferin reporter gene, followed 10 days later by control T cells, BCMA_CAR or BCMA_BCL2L1_CAR T cells IV injection, and despite a skewing to a larger initial disease burden in the BCMA-BCL2L1-CAR group, BCL2L1 armored CAR T cells resulted in more prolonged disease control and animal survival compared to the BCMA_CAR treated mice (FIG 1D). Our studies indicate that BCL2L1 blockade of AICD not only enhanced the viability and proliferation of BCMA targeting CAR T cells but surprisingly also reduced their functional exhaustion. Our findings provide an novel approach for CAR T optimization and overcoming disease relapse resulting from lack of persistence and/or T cells exhaustion. Figure 1 Figure 1. Disclosures Neri: Amgen: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria. Bahlis: Sanofi: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Genentech: Consultancy; Janssen: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; GlaxoSmithKline: Consultancy, Honoraria; BMS/Celgene: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria.

scholarly journals ALLO-819, an Allogeneic Flt3 CAR T Therapy Possessing an Off-Switch for the Treatment of Acute Myeloid Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3335-3335
Author(s):  
Cesar Sommer ◽  
Ivana Djuretic ◽  
Julien Valton ◽  
Duy Nguyen ◽  
Janette Sutton ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Antitumor Efficacy ◽  
Cell Phenotype ◽  
Employment Equity ◽  
Car T Cells ◽  
Car T Cell ◽  
Nsg Mice ◽  
Car T ◽  
Equity Ownership

Abstract Patients with relapsed acute myeloid leukemia (AML) have poor prognosis and limited treatment options. Chimeric antigen receptor (CAR) T cells have demonstrated unprecedented clinical efficacy in hematological malignancies, leading to durable responses in heavily pretreated patients. Adoptive immunotherapies using T cells redirected against AML cells are being pursued as one option with potential curative intent. However, the development of autologous CAR T therapies presents a significant logistical and clinical challenge in a rapidly progressing disease setting such as AML due to the lag time of cell manufacturing. Additionally, harvesting sufficient numbers of healthy T cells from patients with AML may not always be possible. For these reasons the development of an off-the-shelf CAR T cell product may be of benefit. This work details the preclinical evaluation of ALLO-819, an allogeneic CAR T therapy targeting the receptor tyrosine kinase Flt3 (CD135), an AML target with high prevalence in all AML subtypes and limited expression outside of the hematopoietic tissue. To construct a Flt3 CAR, a panel of high affinity (KD values of 0.19 to 233 nM, determined at 37°C) fully-human antibodies was generated using phage display technology. Single-chain variable fragments (scFvs) recognizing different immunoglobulin domains of the extracellular region of Flt3 were inserted into second-generation CAR constructs and tested for their ability to redirect T cell specificity and effector function towards AML cells. A lead CAR exhibiting minimal tonic signaling and potent antitumor activity in orthotopic mouse models of AML (2.5x106 and 1x107 CAR T cells for Eol-1 and Molm-13, respectively) was selected for further engineering to incorporate a safety off-switch in cis. To accomplish this, short amino acid stretches mimicking epitopes for the FDA-approved antibody rituximab were inserted between the hinge and target-binding regions of the CAR. The CAR T cell phenotype and antitumor efficacy were not affected by the presence of the off-switch. In the presence of rituximab, Flt3 CAR T cells were efficiently lysed via complement-dependent cytotoxicity (~ 80 % CAR T cell depletion in 3 hours) in vitro and eliminated in peripheral blood and bone marrow of NSG mice (>100-fold and >300-fold, respectively). Allogeneic ALLO-819 Flt3 CAR T cells with a lower risk of TCR-mediated graft-versus-host disease and resistant to anti-CD52 antibody (alemtuzumab)-mediated lysis were generated by disruption of the T-cell receptor alpha chain (TRAC) and the CD52 loci using TALEN® gene-editing technology. Transient expression of TALEN® in Flt3 CAR T cells resulted in high-efficiency inactivation of both loci and had no impact on T cell phenotype or antitumor efficacy. ALLO-819 Flt3 CAR T cells co-cultured with primary AML blasts ex vivo displayed target-dependent activation, cytokine secretion and cytotoxic activity. Consistent with previous reports, we detected Flt3 expression on a subset of normal hematopoietic stem and progenitor cells (HSPCs) which also showed susceptibility to CAR T cell cytotoxicity. To evaluate off-tumor effects of Flt3 CAR T cells in vivo, NSG mice were administered T cells expressing a CAR with similar affinity to both mouse and human Flt3. Mouse-cross-reactive Flt3 CAR T cells exhibited off-tumor activity that was limited to a subset of bone marrow multipotent progenitors and correlated with antitumor efficacy. Administration of rituximab led to effective depletion of CAR T cells in peripheral blood that was followed by a rapid repopulation of HSPCs to levels observed in naïve mice. In summary, these results support the development of ALLO-819 Flt3 CAR T as a novel immunotherapy for the treatment of AML. Disclosures Sommer: Allogene Therapeutics: Employment, Equity Ownership, Patents & Royalties. Djuretic:Pfizer Inc.: Employment. Valton:Cellectis.Inc: Employment, Equity Ownership, Patents & Royalties. Nguyen:Allogene Therapeutics: Employment, Equity Ownership. Sutton:Allogene Therapeutics: Employment, Equity Ownership. Poulsen:Allogene Therapeutics: Employment, Equity Ownership. Smith:Cellectis. Inc: Employment, Patents & Royalties. Djuretic:Pfizer Inc.: Employment. Chaparro-Riggers:Pfizer Inc.: Employment, Patents & Royalties. Sasu:Allogene Therapeutics: Employment, Equity Ownership, Patents & Royalties.

scholarly journals Infection Temperature Affects the Phenotype and Function of Chimeric Antigen Receptor T Cells Produced via Lentiviral Technology

2021 ◽  
Vol 12 ◽  
Author(s):  
Xin Jin ◽  
Wenyi Lu ◽  
Meng Zhang ◽  
Xia Xiong ◽  
Rui Sun ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
Immune Checkpoints ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
And Function

Chimeric antigen receptor (CAR)-T cell therapy has become an important method for the treatment of hematological tumors. Lentiviruses are commonly used gene transfer vectors for preparing CAR-T cells, and the conditions for preparing CAR-T cells vary greatly. This study reported for the first time the influence of differences in infection temperature on the phenotype and function of produced CAR-T cells. Our results show that infection at 4 degrees produces the highest CAR-positive rate of T cells, infection at 37 degrees produces the fastest proliferation in CAR-T cells, and infection at 32 degrees produces CAR-T cells with the greatest proportion of naive cells and the lowest expression of immune checkpoints. Therefore, infection at 32 degrees is recommended to prepare CAR-T cells. CAR-T cells derived from infection at 32 degrees seem to have a balance between function and phenotype. Importantly, they have increased oncolytic ability. This research will help optimize the generation of CAR-T cells and improve the quality of CAR-T cell products.

CBIO-16. DOSE INTENSIFIED TEMOZOLOMIDE IMPROVES INFILTRATION OF CD70 CAR T CELLS INTO THE GBM TUMOR MICROENVIRONMENT

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi30-vi30
Author(s):  
Aida Karachi ◽  
Jianping Huang ◽  
Haipeng Tao ◽  
Farhad Dastmalchi ◽  
Linchun Jin ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Microenvironment ◽  
T Cell Exhaustion ◽  
Systemic Blood ◽  
Cell Exhaustion ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
And Function

Abstract CD70 CAR T cells developed by our group have anti-tumor efficacy in syngeneic murine GBM model and are currently being developed for first-in-human testing. The objective of this study was to evaluate the impact of temozolomide (TMZ) in various dosing strategies on the expansion, persistence and function of the CD70 CAR T cells after infusion. C57BL/6 mice underwent intracranial implantation of KR-158 overexpressing CD70 tumor cells. Tumor bearing animals were treated with standard dose (SD) (50 mg/kg x 5 days), metronomic dose (MD) (25mg/kg x 10 days), or dose intensified (DI) (75mg/kg x 5 days) of TMZ, followed by 10×106 CAR T cells infusion. Peripheral blood was collected to monitor the persistence of CAR T cells in the systemic blood circulation. Six weeks post treatments, spleens and tumors were collected and CAR T cells abundance and function were measured. TMZ preconditioning resulted in the expected lymphopenia in animals by generation of dose dependent lymphopenia. Circulating CD70 CAR T cells peaked in the systemic blood 2 weeks after infusion (3-fold, p< 0.0001). Markers of T cell exhaustion including PD-1 and TOX expression on CAR T cells were not different between the three TMZ treatment groups compared to control. Six weeks post treatment, CD70 CAR T cells were found to be highly infiltrated within the tumor microenvironment in the DI TMZ group compared to the other groups (4-fold,p < 0.0001). Tumor infiltrating CD70 CAR T cells in the DI TMZ group did not have an increase in PD-1 and TOX expression which was seen in the MD and SD TMZ groups. DI TMZ preconditioning results in greater CD70 CAR T cell trafficking to the tumor without T cell exhaustion compared to lower doses of TMZ. The affects of TMZ on the immune microenvironment to enhance CAR T cell regimens warrants further study.

scholarly journals Identification of dual positive CD19+/CD3+ T cells in a leukapheresis product undergoing CAR transduction: a case report

2020 ◽  
Vol 8 (2) ◽  
pp. e001073
Author(s):  
Liora Schultz ◽  
Shabnum Patel ◽  
Kara Lynn Davis ◽  
Sneha Ramakrishna ◽  
Bita Sahaf ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Population ◽  
Cell Tracking ◽  
Cell Phenotype ◽  
Main Body ◽  
Hematopoietic Stem ◽  
Car T Cell ◽  
Cell Manufacturing ◽  
Car T

BackgroundChimeric antigen receptor (CAR) therapy and hematopoietic stem cell transplantation (HSCT) are therapeutics for relapsed acute lymphocytic leukemia (ALL) that are increasingly being used in tandem. We identified a non-physiologic CD19+/CD3+ T-cell population in the leukapheresis product of a patient undergoing CAR T-cell manufacturing who previously received a haploidentical HSCT, followed by infusion of a genetically engineered T-cell addback product. We confirm and report the origin of these CD19+/CD3+ T cells that have not previously been described in context of CAR T-cell manufacturing. We additionally interrogate the fate of these CD19-expressing cells as they undergo transduction to express CD19-specific CARs.Main bodyWe describe the case of a preteen male with multiply relapsed B-ALL who was treated with sequential cellular therapies. He received an αβ T-cell depleted haploidentical HSCT followed by addback of donor-derived T cells genetically modified with a suicide gene for iCaspase9 and truncated CD19 for cell tracking (RivoCel). He relapsed 6 months following HSCT and underwent leukapheresis and CAR T-cell manufacturing. During manufacturing, we identified an aberrant T-cell population dually expressing CD19 and CD3. We hypothesized that these cells were RivoCel cells and confirmed using flow cytometry and PCR that the identified cells were in fact RivoCel cells and were eliminated with iCaspase9 activation. We additionally tracked these cells through CD19-specific CAR transduction and notably did not detect T cells dually positive for CD19 and CD19-directed CARs. The most likely rationale for this is in vitro fratricide of the CD19+ ‘artificial’ T-cell population by the CD19-specific CAR+ T cells in culture.ConclusionsWe report the identification of CD19+/CD3+ cells in an apheresis product undergoing CAR transduction derived from a patient previously treated with a haploidentical transplant followed by RivoCel addback. We aim to bring attention to this cell phenotype that may be recognized with greater frequency as CAR therapy and engineered αβhaplo-HSCT are increasingly coupled. We additionally suggest consideration towards using alternative markers to CD19 as a synthetic identifier for post-transplant addback products, as CD19-expression on effector T cells may complicate subsequent treatment using CD19-directed therapy.

scholarly journals IL-15 Enhances the Persistence and Function of BCMA-targeting CAR-T Cells Compared to IL-2 or IL-15/IL-7 by Limiting CAR-T Cell Dysfunction and Differentiation

Cancers ◽  
2021 ◽  
Vol 13 (14) ◽  
pp. 3534
Author(s):  
Anthony M. Battram ◽  
Mireia Bachiller ◽  
Victor Lopez ◽  
Carlos Fernández de Larrea ◽  
Alvaro Urbano-Ispizua ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Dysfunction ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
T Cell Dysfunction ◽  
And Function

Chimeric antigen receptor (CAR)-T cell immunotherapy has revolutionized the treatment of B-lymphoid malignancies. For multiple myeloma (MM), B-cell maturation antigen (BCMA)-targeted CAR-T cells have achieved outstanding complete response rates, but unfortunately, patients often relapse within a year of receiving the therapy. Increased persistence and reduced dysfunction are crucial features that enhance the durability of CAR-T cell responses. One of the factors that influence CAR-T cell in vivo longevity and loss of function, but which has not yet been extensively studied for BCMA-directed CAR-T cells, are the cytokines used during their production. We here compared the impact of IL-2, IL-15 and a combination of IL-15/IL-7 on the phenotype and function of ARI2h, an academic BCMA-directed CAR-T cell that is currently being administered to MM patients. For this study, flow cytometry, in vitro cytotoxicity assays and analysis of cytokine release were performed. In addition, ARI2h cells expanded with IL-2, IL-15, or IL-15/IL-7 were injected into MM tumor-bearing mice to assess their in vivo efficacy. We demonstrated that each of the cytokine conditions was suitable for the expansion of ARI2h cells, with clear in vitro activity. Strikingly, however, IL-15-produced ARI2h cells had improved in vivo efficacy and persistence. When explored further, it was found that IL-15 drove a less-differentiated ARI2h phenotype, ameliorated parameters related to CAR-T cell dysfunction, and lowered the release of cytokines potentially involved in cytokine release syndrome and MM progression. Moreover, we observed that IL-15 was less potent in inducing T cell senescence and DNA damage accumulation, both of which may contribute to an unfavorable CAR-T cell phenotype. These findings show the superiority of IL-15 to IL-2 and IL-15/IL-7 in the quality of anti-BCMA CAR-T cells, particularly their efficacy and persistence, and as such, could improve the duration of responses if applied to the clinical production of CAR-T cells for patients.

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